1. Field of the Invention
This invention relates to immunogen conjugates comprising digoxigenin derivatives, particularly carboxy- and amino-functionalized derivatives coupled to conventional immunogenic carrier materials, and anti-digoxin antibodies prepared against such immunogen conjugates. Such antibodies are useful in immunoassays for determining digoxin in biological fluids. The invention also relates to labeled digoxigenin conjugates useful in particularly preferred immunoassay techniques.
2. Background of the Invention
It is of established medical value to monitor the concentration of digoxin in the bloodstream of patients under treatment with the drug. Immunoassays are the currently most common methods used to determine digoxin in blood samples, e.g., serum or plasma, and are based on the specific binding of anti-digoxin antibodies to the drug in the test sample.
Digoxin is a cardiac glycoside consisting of an aglycone, digoxigenin, and three glycosidic digitoxose residues linked to the aglycone at the C-3 position. The glycoside is a hapten, that is, it is incapable of stimulating antibody production unless it is injected into the host animal in the form of a conjugate with an immunogenic carrier material, e.g., a protein such as albumin from an animal of a different species. The conventional immunogen conjugate for preparing anti-digoxin antibodies comprises the glycoside chemically linked through a modification of the terminal glycosidic residue to a carrier. The terminal residue is oxidized with periodate to open the ring and form a dialdehyde derivative which is readily couplable to free amino groups in the carrier [Butler and Chen, Proc. Natl. Acad. Sci. USA 57:71(1967), and Smith et al, Biochem 9:331(1970)]. The result is a digoxin-carrier conjugate.
Antibodies against the related cardiac glycoside digitoxin have been prepared from a conjugate of the aglycone coupled at the C-3 position to the carrier. The aglycone 3-o-succinoyl-digitoxigenin has been coupled by conventional techniques to protein carriers [Oliver et al, J. Clin. Invest. 47:1035(1968)].
3-Substituted digoxigenin and digitoxigenin derivatives have been proposed and used for the purpose of preparing labeled conjugates to be used in conjunction with anti-digoxin and anti-digitoxin antibodies in performing immunoassays. Representative of this art are U.S. Pat. Nos. 3,981,982 and 4,064,227 and U.S. Pat. Nos. 4,039,385; 4,213,893; and 4,273,866 relating to various nonradioisotopically labeled conjugates.
U.S. Pat. Nos. 4,217,280 and 4,219,549 describe the synthesis of various 3-amino-3-desoxydigoxigenin derivatives and their use in cardiotonic therapy. The preparation of 3-digoxigenone is known from Tamm and Gubler, Helv. Chim. Acta 42:239(1959) and Shimizu and Mituhashi, Tetrahedron 24:4207(1968). Boutique and Koenig, Bull. Chem. Soc. Fr. 1973(2), part 2, 750 describe the reductive amination of steroidal ketones. The use of sodium cyanoborohydride in the reductive amination of aldehydes and ketones is described by Borch et al, J. Amer. Chem. Soc. 93:2987(1971). 3-Aminocardenolides were prepared by Hanser et al, Helv. Chim. Acta 56(8):2782(1973).